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Review

 
Review Mitochondria as a central sensor for axonal degenerative stimuli Felipe A. Court and Michael P.fcourt@bio.puc.cl ); Coleman, M.P. ( michael.coleman@babraham.ac.uk ). Keywords: axonal degeneration; mitochondria; axonal transport; mitochondrial permeability transition pore; reactive oxygen species. many cell types. Because of this discontinuity, the delivery of material and exchange with other mitochondria may be more dependent on mechanisms such as fusion and fission. Third, their transport has to be carefully regulated to ensure that mitochondria are focused in the correct regions of the huge axonal compartment [4] , which can be several hundred times larger than neuronal soma. In this way, high requirements for adenosine triphosphate (ATP) syn- thesis and Ca buffering can be met. This review considers the extent to which unique fea- tures of axonal mitochondria underlie axon-specific degen- eration mechanisms, making them a nodal point for decisions on axon survival. In some cases, axons degener- ate through failure to maintain a healthy mitochondrial population at literally ‘arm’s length’ from the cell body, whereas in others mitochondria may play a more active role in axon degeneration. The latter process may be much faster, whereas a steady decline in mitochondria quality in axons may take many years and only occur when axonal transport further declines with age. Challenges for axonal mitochondria Mitochondrial defects are surprisingly prevalent in axon degeneration disorders ( Table 1 ). It is interesting to con- sider whether this reflects properties of mitochondria that are important within axons in particular and how such properties may fit together. Mitochondrial fusion and fission in neurodegenerative conditions Mitochondrial fusion and fission are particularly promi- nent among the functions of the proteins in Table 1 . Proteins controlling these processes are mutated in subtypes of peripheral neuropathy, optic atrophy and he- reditary spastic paraplegia (HSP). Mitofusin 2, an outer mitochondrial membrane protein mediating fusion is mu- tated in the pure axonal disorder Charcot-Marie-Tooth Type 2A [5] , whereas optic atrophy 1 (OPA1), which has a related role in fusing the inner mitochondrial membrane, is defective in autosomal dominant optic atrophy [6] . Para- plegin (encoded by the SPG7 gene), which is mutated in some types of HSP, regulates control of mitochondrial size by OPA1 [7] and its absence results in giant mitochon- dria [8] . Ganglioside-induced differentiation-associated protein-1 (GDAP1), a protein that enhances mitochondria fragmentation, is mutated in the mixed axon/myelin dis- order Charcot-Marie-Tooth Disease type 4A (CMT4A) [9] . 364 0166-2236/$ – see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tins.2012.04.001 Trends in Neurosciences, June 2012, Vol. 35, No. 6 " id="pdf-obj-0-6" src="pdf-obj-0-6.jpg">

Mitochondria as a central sensor for axonal degenerative stimuli

Felipe A. Court 1 , 2 and Michael P. Coleman 3

1 Millennium Nucleus for Regenerative Biology, Faculty of Biology, Catholic University of Chile, Santiago 8331150, Chile 2 Neurounion Biomedical Foundation, Santiago, Chile 3 Signalling Programme, The Babraham Institute, Babraham Research Campus, Babraham, Cambridge CB22 3AT, UK

Axonal degeneration is a major contributor to neuronal dysfunction in many neurological conditions and has additional roles in development. It can be triggered by divergent stimuli including mechanical, metabolic, infec- tious, toxic, hereditary and inflammatory stresses. Axo- nal mitochondria are an important convergence point as regulators of bioenergetic metabolism, reactive oxygen species (ROS), Ca 2+ homeostasis and protease activa- tion. The challenges likely to render axonal mitochondria more vulnerable than their cellular counterparts are reviewed, including axonal transport, replenishing nuclear-encoded proteins and maintenance of quality control, fusion and fission in locations remote from the cell body. The potential for mitochondria to act as a decision node in axon loss is considered, highlighting the need to understand the biology of axonal mitochon- dria and their contributions to degenerative mecha- nisms for novel therapeutic strategies.

Introduction

Neuronal soma and axons die by very different mecha- nisms. Axon degeneration is in many cases an autonomous process that is distinct from classical apoptosis but can be genetically regulated. Conversely, a protein that protects axons, the slow Wallerian degeneration protein (Wld S ), has no known effect on survival of the soma [1,2]. As molecular mechanisms underlying axon survival and degeneration become better understood, it is important to ask what makes them specific for axons. Many mitochondrial dysfunctions lead to disorders in which axon degeneration is the predominant feature, or a prominent early step (Table 1). Thus, axons may face a greater challenge than neuronal soma or dendrites in maintaining sufficient functioning of mitochondria for sur- vival. Mitochondria, long known to have multiple roles in cell death, differ strikingly between axons and soma. First, their elongated shape, at least in peripheral nerve axons, is specialized for life in a long narrow cylinder [3]. Mainte- nance of this shape is critical for efficient delivery by axonal transport so that even moderate swelling will block both their own transport and that of many other cargoes (Figure 1). Second, axonal mitochondria are discrete struc- tures, in contrast to the syncytia more typically seen in

Corresponding authors: Court, F.A. (fcourt@bio.puc.cl); Coleman, M.P. (michael.coleman@babraham.ac.uk). Keywords: axonal degeneration; mitochondria; axonal transport; mitochondrial permeability transition pore; reactive oxygen species.

many cell types. Because of this discontinuity, the delivery of material and exchange with other mitochondria may be more dependent on mechanisms such as fusion and fission. Third, their transport has to be carefully regulated to ensure that mitochondria are focused in the correct regions of the huge axonal compartment [4], which can be several hundred times larger than neuronal soma. In this way, high requirements for adenosine triphosphate (ATP) syn- thesis and Ca 2+ buffering can be met. This review considers the extent to which unique fea- tures of axonal mitochondria underlie axon-specific degen- eration mechanisms, making them a nodal point for decisions on axon survival. In some cases, axons degener- ate through failure to maintain a healthy mitochondrial population at literally ‘arm’s length’ from the cell body, whereas in others mitochondria may play a more active role in axon degeneration. The latter process may be much faster, whereas a steady decline in mitochondria quality in axons may take many years and only occur when axonal transport further declines with age.

Challenges for axonal mitochondria

Mitochondrial defects are surprisingly prevalent in axon degeneration disorders (Table 1). It is interesting to con- sider whether this reflects properties of mitochondria that are important within axons in particular and how such properties may fit together.

Mitochondrial fusion and fission in neurodegenerative conditions Mitochondrial fusion and fission are particularly promi- nent among the functions of the proteins in Table 1. Proteins controlling these processes are mutated in subtypes of peripheral neuropathy, optic atrophy and he- reditary spastic paraplegia (HSP). Mitofusin 2, an outer mitochondrial membrane protein mediating fusion is mu- tated in the pure axonal disorder Charcot-Marie-Tooth Type 2A [5], whereas optic atrophy 1 (OPA1), which has a related role in fusing the inner mitochondrial membrane, is defective in autosomal dominant optic atrophy [6]. Para- plegin (encoded by the SPG7 gene), which is mutated in some types of HSP, regulates control of mitochondrial size by OPA1 [7] and its absence results in giant mitochon- dria [8]. Ganglioside-induced differentiation-associated protein-1 (GDAP1), a protein that enhances mitochondria fragmentation, is mutated in the mixed axon/myelin dis- order Charcot-Marie-Tooth Disease type 4A (CMT4A) [9].

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Table 1. Mitochondrial proteins mutated in disorders with prominent axonal degeneration a

Disease

Protein

Disease type

Possible protein function

Refs

Charcot-Marie-Tooth

Mitofusin 2

CMT2A

Outer mitochondrial membrane protein ER protein Axonal mitochondrial transport ER-mitochondrial tethering

[5,32,103]

HSPB1 (HSP27)

CMT2F

Molecular chaperone Neurofilament organization Microtubule binding

[104,105]

HSPB8 (HSP22)

CMT2L

Molecular chaperone Associated with autophagy

[106]

Gdap1

CMT4A

Outer mitochondrial membrane protein Mitochondrial fission

[9]

Friedrich’s ataxia

Frataxin

Friedrich’s ataxia

Mitochondrial matrix iron chaperone Redox regulation

[22]

Hereditary spastic

Paraplegin

SPG7

Mitochondrial ATPase

[8]

paraplegia

Hsp60

SPG13

Mitochondrial chaperone Resistance to oxidative stress (?)

[107]

Spartin

SPG20

Several protein partners, localization and functions Required for mitochondrial calcium uptake capacity

[81]

Reep1

SPG38

Binds spastin Associates with microtubules ER-shaping and mitochondrial dynamics (?)

[108]

Optic atrophy/neuropathy

OPA1

Optic atrophy

Inner mitochondrial membrane protein Mitochondrial fusion

[6,109]

Complex 1 subunits

Leber’s hereditary

Complex I subunit of mitochondria

[110]

ND1, 4, 6

optic neuropathy

Decrease ATP production

Parkinson’s disease

Parkin

PARK2

Cytosolic E3 ubiquitin ligase Mitochondrial quality control Microtubule dynamics

[18]

PINK1

PARK6

Mitochondrial quality control

[17]

DJ-1

PARK7

Atypical peroxidase Regulation of oxidant defenses

[111,112]

LRRK2

PARK8

Partial mitochondrial localization

[113]

HTRA2

PARK13

Mitochondrial intermembrane space Pro-apoptotic

[114]

POLG1

Parkinson’s disease,

Inner mitochondrial membrane

[115]

Alper’s syndrome

Mitochondrial DNA synthesis, replication and repair

Sensory neuropathy

Bcl-w

Small fiber sensory neuropathy

Mitochondrial localization Anti apoptotic Bcl-2 family member

[67]

a Abbreviations: HTRA2, high temperature requirement protein 2; LRRK2, leucine-rich repeat kinase 2; POLG1, mitochondrial DNA polymerase gamma 1; Reep1, receptor expression enhancing protein 1.

The GTPase dynamin-related protein 1 (DRP1), in addition to being associated with mitochondrial fission, is also required for neurite development or maintenance in pri- mary culture [10]. In humans, a mutation in DRP1 has been associated with severe and lethal defects in the nervous system [11]. Fusion and fission of axonal mito- chondria may also be a primary target in toxic models, as chronic exposure to low levels of rotenone causes loss of dopaminergic neuronal processes without cell death in a process that may require mitochondrial fission [12].

Mitochondria quality control and neuronal degeneration

Quality control of mitochondria is another emerging theme, especially in Parkinson’s disease (PD), where a growing body of evidence points to a ‘dying back’ process of early axon and synapse loss [13]. Experiments in non-neuronal cell culture suggest that accumulation of phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)

on the surface of dysfunctional mitochondria acts as a signal for their removal by mitophagy [14,15]. By contrast, regular voltage-dependent proteolysis of PINK1 on healthy mito- chondria maintains low levels of this protein [14]. This

degradation stops if mitochondrial membrane potential falls, causing rapid accumulation of PINK1 and recruitment of Parkin leading to mitophagy. Recent data confirm that Parkin recruitment occurs in neuronal soma [16]. Genetic evidence supports a similar mechanism in vivo as loss-of- function mutations in PINK1 or Parkin cause PD [17,18] and Drosophila studies indicate that Parkin lies down- stream of PINK1 [19]. Quality control becomes particularly intriguing in the context of the huge axonal arbors of dopaminergic neurons in mammalian brain [20]. Degeneration of these nerve terminals clearly precedes death of the soma [13] so any failure of quality control may occur here first. In distal axons of sensory neurons, mitochondria fragments are engulfed by microtubule-associated protein 1A/1B-light chain 3 (LC3)-positive vesicles and the resulting autopha- gosomes fuse with lysosomes as they move retrogradely [21]. The importance of retrograde transport is consistent with the accumulation of depolarized mitochondria in distal axons in a Drosophila model of Friedreich ataxia, associated with a failure of retrograde axonal transport [22]. Whether these first stages of mitophagy in axons use

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* * Injured axon Healthy axon Healthy axon (d) (c) Pathological (HSP) (a) (b) TRENDS in
*
*
Injured axon
Healthy axon
Healthy axon
(d)
(c)
Pathological (HSP)
(a)
(b)
TRENDS in Neurosciences

Figure 1. Mitochondrial morphology in healthy and degenerating axons. (a) Healthy axonal mitochondria (depicted by the arrow) are often elongated discrete structures that are oriented along the axon shaft, as shown in this electron micrograph of mouse optic nerve. Scale bar, 2 mm. Reproduced, with permission, from [102]. (b) Healthy mitochondria (arrows) in axons from mouse optic nerves occupy a small fraction of axonal diameter but after nerve injury (c) mitochondria (*) swell to dimensions likely to perturb axonal transport of both themselves and other cargoes. Scale bars in (b) and (c), 300 nm. Reprinted, with permission, from [60]. (d) Similar morphologies occur in axonal pathology, such as in the spinal cord of SPG7-deficient mice, which model hereditary spastic paraplegia (HSP). Scale bar, 800 nm. Reproduced, with permission, from [8].

the PINK1/Parkin mechanism remains to be clarified. Both proteins are present in axons, where they downreg- ulate axonal transport of mitochondria [16,23] and Parkin recruitment can occur in neurites when mitochondria transport is blocked [16]. However, clear evidence for axo- nal Parkin recruitment in more physiological conditions has not yet been reported. Whatever their identity, the proteins targeting mitochondria for mitophagy in distal axons must be delivered by anterograde transport, so this transport is important for maintaining a pool of healthy axonal mitochondria.

Mitochondrial transport in large neuronal compartments In addition to retrograde trafficking of autophagosomes, and anterograde transport of proteins for mitophagy, axo- nal transport plays a third critical role in maintaining axonal mitochondria. Unlike their somatic counterparts, 10–30% of axonal mitochondria are trafficked over huge distances [3]. While the soma is the site where some axonal mitochondria are generated [24], mitochondrial biogenesis also occurs within isolated axons [25]. The need to traffic mitochondria from the soma when axons can generate their own probably reflects the fact that mitochondrial DNA encodes only 13 out of hundreds of mitochondrial proteins [26]. Nuclear-encoded proteins need to be deliv- ered to replace natural turnover. One attractive model is that motile mitochondria, which are usually smaller [3], deliver these nuclear-encoded proteins into axons, where they fuse with large, stationary mitochondria to replenish them (Figure 2), although local protein synthesis [27] and local mitochondrial import of

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nuclear-encoded proteins may also contribute to this deliv- ery. According to this model, disruption of this anterograde trafficking should result in gradual deterioration of axonal mitochondria. It is interesting that such disruption has been observed to occur in animal models of familial amyo- trophic lateral sclerosis (ALS) [28], tauopathy [29,30], HSP [31], CMT2A [32,33], Alzheimer’s disease (AD) [34], Huntington’s disease (HD) [35] and toxicity by A beta or 1-methyl-4-phenylpyridinium ion (MPP + ) [36–38]. Some studies report that axonal transport of mitochondria can be separated from pathogenesis in mouse models of ALS [39,40] but a contributory role to some axonal disorders does seem likely [41]. This could be particularly true in age- related disorders, as transport decline during normal age- ing [30] could render older axons less tolerant of any additional transport impairment. Anterograde transport, fusion, fission and quality con- trol are likely to act together to maintain the functionality of axonal mitochondria and long-term axon survival (Figure 2). In this model, stationary mitochondria are regularly serviced by delivery and fusion of small mobile mitochondria carrying newly-synthesized nuclear-encoded proteins. Consistent with this, mitochondrial flux increases when nuclear-encoded mitochondrial DNA poly- merase Pol gamma is deleted, as though the axon were trying to compensate for the shortage [42]. Both fusion and fission of mitochondria occur in axons [12] and asymmetric fission has been shown in other cell types, producing daughter mitochondria with differing membrane poten- tials [43]. This should allow less competent mitochondria to be removed by quality control, potentially involving PINK1/Parkin. Defects in the anterograde transport of

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Axon Distal Soma Proximal
Axon
Distal
Soma
Proximal
PINK1? Fission Fusion replenishes Stationary, old Moving, new Mitophagy (a) (b) (c) TRENDS in Neurosciences
PINK1?
Fission
Fusion replenishes
Stationary, old
Moving, new
Mitophagy
(a)
(b)
(c)
TRENDS in Neurosciences

Figure 2. Proposed model for quality control of axonal mitochondria. (a) A small anterogradely moving mitochondrion [3] (blue) is proposed to contain new nuclear- encoded proteins consistent with biogenesis of some mitochondria within the cell body [24]. By contrast, the larger, stationary mitochondrion (red) is proposed to contain older proteins that have resided in the axon for some time. (b) Fusion of moving mitochondrion to stationary mitochondrion [12] mixes their contents (purple). (c) A stationary mitochondrion is depicted as undergoing asymmetric fission [43]. The smaller daughter mitochondrion (red) is targeted for mitophagy within the axon [21], potentially involving axonally localized PINK1 [23], and retrogradely trafficked for lysosomal degradation [16,21]. The large stationary mitochondrion is replenished by the delivery of new proteins and disposal of less functional material. In this model, axonal transport, mitochondrial fusion and fission, and signaling proteins important for mitophagy are all required to maintain functionality of the axonal mitochondria population.

mitochondria themselves, or of proteins needed for mito- phagy, and defects in fusion or fission or mitophagy would all disrupt this mechanism. Consistent with this, there are indications that mitochondrial fusion and fission are dis- rupted in HD [35,44] and AD [34,45]. In addition, short fragmented mitochondria accumulate in dystrophic axons close to amyloid plaques [46], whereas a-synuclein also stimulates mitochondrial fission [47] and defects in autop- hagy lead to severe swelling in Purkinje cell axons [48,49]. Finally, complex IV negative mitochondria accumulate in axons of multiple sclerosis (MS) patients as though quality control has failed at some level [50].

Participation of mitochondria in Wallerian degeneration

Recent data suggest that mitochondria have important roles in one specific axon degeneration mechanism known as Wallerian degeneration (WD). WD is triggered by mechani- cal disconnection of axons from cell bodies and serves as a

model for axon degeneration when axonal transport is dis- rupted [51]. The supporting evidence is that an aberrant protein generated by a spontaneous mutation, Wld S , is able to delay axon degeneration both after injury and in some axonal transport disorders, genetically linking these mech- anisms [52]. It has been demonstrated that several parallels exists between axonal degeneration triggered by mechanical injury (i.e. WD) and the axonal degenerative events acti- vated by some toxic, inflammatory or metabolic stressors in diverse neurodegenerative conditions [53]. Therefore, WD represents an experimental model extensively used to define the mechanisms involved in axonal degeneration. After mechanical nerve injury, axons exhibit a latent phase in which the structures remain apparently normal

(1–2 days in laboratory animals), followed by a cata- strophic phase in which all axonal structures collapse rapidly, in a well-ordered sequence [51]. With a few excep- tions, collapse of severed axons occurs in animals of widely separated phyla [51]. Agents that interfere with microtu- bular function also trigger degeneration, again pointing to a role for disruption of microtubule-assisted transport in axonal degeneration [51]. Interestingly, several links have emerged between WD and mitochondria. Overexpression of the mitochondrial NAD + synthesizing enzyme nicotinamide mononucleo- tide adenylyltransferase 3 (NMNAT3) delays axonal degeneration [54]. Furthermore, the WD-protective Wld S protein, which is a chimera between nuclear NMNAT1 and a non-catalytic sequence from a ubiquitin ligase, is also partially localized to axonal mitochondria [54,55]. The enzymatic activity of NMNAT3 or Wld S is required for their protective action. Although this was originally thought to implicate NAD + in axonal degeneration, it is possible that another metabolite handled by these enzymes has a critical role [56]. Thus, localization of Wld S and NMNAT3 in axonal mitochondria could be an important requirement for the protective effect, but because neither is confined to mitochondria this still requires confirmation. Although the protective mechanism of overexpressed NMNATs has not yet been elucidated, the involvement of NAD + -dependent pathways in mitochondrial energy metabolism and redox capacity in the cytoplasm or mito- chondria [57] suggest a mechanism linking loss of mito- chondrial function to axonal degeneration [58]. For example, this could occur when one or more NMNATs are not properly trafficked to axons [59].

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Recent data indicate a direct association between WD and activation of the mitochondrial permeability transition pore (mPTP). Genetic or pharmacologic inhibition of mPTP activation delays axonal degeneration downstream Wld S protection [60]. The mPTP has been functionally associat- ed to several neurodegenerative conditions harboring axo- nal degeneration, including MS, AD, ALS and cerebral ischemia [61–64]. Interestingly, two of the main activators of the mPTP in excitable cells, Ca 2+ and ROS [65], are pathways experimentally defined as participants in the axonal degeneration program. Moreover, a recent report shows that Wld S enhances basal mitochondrial motility and increases Ca 2+ buffering capability of axonal mito- chondria [66], consistent with the involvement of mPTP in axonal degeneration [60]. The mPTP is molecularly distinct from mitochondrial- dependent apoptosis. This is important because classical apoptosis involving B cell lymphoma 2 (Bcl-2)-associated X protein (Bax), BCL2-homologous antagonist/killer (Bak), or Bcl-2, is not required for WD [51]. Therefore, activation of the mPTP could be part of an axonal-specific program of degeneration that reflects specific axonal characteristics such as distance from the nucleus and unique require- ments of axonal mitochondria, in contrast to classic apo- ptosis taking place in the neuronal soma. Interestingly, a recent report suggests an axon-specific role also for the mitochondrial anti apoptotic protein Bcl-w (also known as Bcl-2L2), at least in a subset of sensory axons [67]. Thus, there may be several pathways in which mitochondria contribute to the specific degeneration of axons.

Mitochondrial changes after degenerative stimuli Axonal degeneration after heterogeneous induction stimuli is considered to proceed through a Wallerian-like mechanism if it is genetically delayed by Wld S [51]. Thus, diverse activators converge onto a common degeneration pathway, suggesting one or more integration points. Axo- nal mitochondria appear to be one such integration point, where pro-degenerative stimuli combine to produce the appropriate survival or degeneration response. Features of mitochondrial dysfunction occur in many disorders, including Ca 2+ dyshomeostasis, ATP depletion, ROS gen- eration and mitochondrial swelling, suggesting mitochon- dria act as a central sensor for degenerative stimuli. According to this model, degradative processes should be activated when concerted stimuli surpass the mitochon- drial homeostatic capacity. Mitochondria control dynamic changes in the cyto- plasmic levels of Ca 2+ , ATP and ROS, and these cellular pathways are connected by several feedback loops [68]. Stimuli affecting one or more of these components are integrated by axonal mitochondria and if a new homeo- static state is not reached, ATP levels decline, intracellular Ca 2+ levels increase and ROS is produced, which culminate in the activation of positive feedback loops that target axons for destruction. Importantly, the sensitivity of mito- chondria to diverse stimuli will depend on genetic and environmental factors, including the cell- and tissue- specific context. For example, mutations that change mitochondrial parameters such as trafficking, respiratory activity, Ca 2+ buffering or ROS generation constitute

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genetic risk factors that increase axonal vulnerability to sublethal stimuli (Table 1). Several mitochondrial proper- ties, including ATP production, Ca 2+ uptake, buffering and release, exhibit regional, cellular and subcellular varia- tions in the nervous system [69]. Mitochondria in axons with unique morphology and metabolic requirements, such as the huge axonal arbors of dopaminergic neurons or meter long peripheral nerve axons, are likely to differ in susceptibility to stressors compared to somatic ones (see above). Environmental factors including caloric intake and toxins such as heavy metals modify the strength of feed- back loops between Ca 2+ , ATP and ROS [68]. Finally, aging, a well-established risk factor for neurodegenerative conditions [70], profoundly affects the homeostasis of the Ca 2+ /ATP/ROS triad, especially in excitable cells, with a decrease in axonal transport in aging axons as one con- tributing factor [30].

Energetic metabolism and ionic homeostasis in axons Excitability comes at a high energetic price. Maintaining ionic gradients and action potential propagation use most cellular energy, exceeding the demands of associated glia by fourfold [71]. Mitochondrial ATP production provides most axonal energy, and ATP consumption is central to mitochondrial homeostatic capacities regulating Ca 2+ and ROS [68]. Energy metabolism, therefore, is a key parame- ter in axons. Mutations in mitochondrial DNA (mtDNA) affecting the electron transport chain, such as Leber’s Hereditary Optic Neuropathy (LHON), reduce ATP pro- duction and increase ROS generation leading to optic nerve degeneration ([72] and Table 1) and decreased ATP pro- duction is also associated with reduced mitochondrial transport and dying-back neuropathy in Friedreich ataxia [22]. Neuronal activity consumes ATP and high frequency stimulation leads to axonal degeneration [73] and neuronal hyperexcitability or ion channel mutations leading to per- sistent currents are associated to axonopathies and neu- ronal cell death [74–76]. When the energy supply fails in axons, Na + -K + ATPase in membranes stop working, causing intra-axonal Na + accumulation, reversal of the Na + -Ca 2+ exchanger and increase in Ca 2+ [77]. Normally, mitochondria face regular increases in Ca 2+ concentration, which is integrated in a homeostatic mechanism regulating mitochondrial trans- port and ATP production [4,78] but reduced Ca 2+ handling underlies neuronal degeneration in several diseases [79]. Mutant huntingtin-expressing neurons show enhanced Ca 2+ sensitivity and reduced mitochondrial Ca 2+ uptake [80], and in a form of HSP caused by a mutation in the spartin (SPG20) gene, mitochondria show a decreased membrane potential and reduced Ca 2+ buffering [81].

Oxidative stress as a degenerative intermediate in axons

On the dark side of the mitochondrial triad is ROS, histori- cally considered a damaging by product of respiration, but recently shown to have physiological signaling functions

[82]. Excess ROS becomes deleterious and axons seem particularly sensitive [38]. Consequently, several neurode- generative conditions are associated with increased ROS [83]. Increase in axonal ROS and axonal degeneration trig- gered by the mitochondrial electron transport inhibitor

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rotenone or by exogenous oxidants is prevented by NMNAT3 overexpression; and vincristine, which causes axonal degeneration, leads to increase in ROS, which is also prevented by NMNAT3 overexpression [84]. Cellular antioxidative mechanisms including superoxide dismutases (SODs) and glutathione generating enzymes normally keep ROS at a low level [85] and genetic deletion of the antioxidant enzyme SOD-1, which is localized to axonal mitochondria in addition to other neuronal domains, triggers axonal degeneration in vitro [86]. Taken together, these findings point to an important role of oxidative stress in an as yet undefined step of the axonal degeneration program. One possibility is that overexpressed NMNATs, either within or associated with mitochondria, could exert their protective function by blocking or reducing ROS pro- duced by very diverse pro-degenerative stimuli. A recent report demonstrates that Wld S also protects from diabetes- induced peripheral neuropathies and retinal ganglion cell degeneration, an effect that might be related to reduction in oxidative stress and energy depletion [87]. ROS generation occurs in virtually all conditions involv- ing microglia and astrocyte activation or macrophage in- filtration, common features of most neurodegenerative diseases [88]. Nitric oxide (NO) induces axonal degenera- tion, especially in electrically active axons [89], an example of the synergistic induction of axonal degeneration. In the experimental autoimmune encephalomyelitis (EAE) model of MS, characterized by axonal degeneration, inflammato- ry episodes lead to elevation of NO by T-cell-activated macrophages, inhibiting mitochondrial respiration and ATP production, and inducing free radical production [88]. Loss of mitochondrial SOD-2 in damaged motor axons accelerates axonal degeneration [90], suggesting that after injury antioxidant enzymes control excess ROS.

Integration by mitochondria: the Ca 2+ /ATP/ROS triad out of control Once intra-axonal homeostatic capability is exceeded, mi- tochondria are confronted by high Ca 2+ and ROS levels,

ideal conditions to activate mitochondrial cell death pro- cesses (Figure 3). Poor quality control of mitochondria (above) is likely to reduce this capability. In these condi- tions, mitochondrial dysfunction and mPTP activation would produce a catastrophic resolution of pro-degenera- tive stimuli, most likely executed by Ca 2+ -dependent cal- pain activation [91]. Importantly, the threshold for mPTP activation decreases with age [92,93], a likely underlying factor in age-related axonal degeneration found in several neurodegenerative conditions, such as AD [70]. mPTP sensitivity is also tissue dependent [94] and neuronal mitochondria are more sensitive to Ca 2+ overload and opening of the mPTP than astrocytic mitochondria [95,96]. Whether diverse neuronal populations also differ in sensitivity to mPTP induction, and hence, in their susceptibility to genetic and toxic insults, remains to be investigated.

Axonal endoplasmic reticulum

Finally, other organelles are likely to participate in a concerted program that ultimately leads to axonal demise. In axons, the endoplasmic reticulum (ER) forms an extend- ed network in close association with the plasmalemma and mitochondria [97]. ROS and ATP also modulate extra- mitochondrial Ca 2+ pools; ROS targets the ryanodine re- ceptor in the ER, enhancing Ca 2+ release [98]; Ca 2+ uptake into the ER by the sarcoplasmic-endoplasmic reticulum Ca 2 -ATPase (SERCA) pump is ATP-dependent. Therefore, intra-axonal Ca 2+ homeostasis is probably jointly regulat- ed by ER and mitochondria [99], and the close apposition of the ER to the axonal plasma membrane might provide a structural support to achieve Ca 2+ control [77]. Interest- ingly, mitofusin-2, which is mutated in the pure axonal disorder CMT2A with mitochondrial transport defects [33], mediates ER-mitochondrial tethering [100], which may be important for both Ca 2+ handling and mitochondrial trans- port. It is also worth noting that a recent study has suggested that ER tubules play an important role in defining the position of mitochondrial division sites

Axon TRENDS in Neurosciences ? Ca 2+ ROS Axonal degeneration Calpain activation ATP Ca 2+ Glia
Axon
TRENDS in Neurosciences
?
Ca 2+
ROS
Axonal degeneration
Calpain activation
ATP
Ca 2+
Glia
Ca 2+
MITO
mPTP
VDAC+uniporter
Glia
Axon
Soma

Figure 3. The contribution of axonal mitochondria in axonal degeneration. A simplified scheme of the factors directly or indirectly controlled by mitochondria that might contribute to axonal degeneration. As shown in the lower diagram, representing a segment of the axon, mitochondria (MITO) physiologically interact with interconnected pathways (black arrows) that regulate energetic metabolism, Ca 2+ homeostasis and ROS generation. High Ca 2+ in the mitochondria, channeled from the axonal cytoplasm by the voltage-dependent anion channel (VDAC) and the uniporter, together with elevated ROS levels, constitute ideal conditions to activate the mitochondrial permeability transition pore (mPTP). When the energy supply fails in axons, Ca 2+ influx through neuronal Na + -Ca 2+ exchangers, as well as by voltage dependent Ca 2+ channels, will contribute to Ca 2+ increase in the axonal cytoplasm (dashed red arrows). Oxidative damage to axonal proteins and lipids together with Ca 2+ -activated calpains probably constitute the point of no return for axonal destruction (red arrows).

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[101]. Although this study was not performed in neurons, it is interesting to speculate that mitochondrial fission with- in axons may be influenced by the ER in a similar manner.

Concluding remarks and future perspectives

Genetic as well as correlative data suggest that mitochon- drial dysfunction is tightly associated with several neurodegenerative conditions characterized by axonal degeneration. The unique morphological and functional features of axons impose a challenge for mechanisms of mitochondrial transport and quality control, increasing susceptibility of axonal mitochondria to genetic and toxic insults compared to their somatic counterparts. Mitochon- dria regulate energetic metabolism, Ca 2+ homeostasis and ROS generation, which are interconnected pathways that will respond to a variety of noxious stimuli resulting in homeostatic control or axonal destruction (Figure 3). A pathway involved in many cellular processes is a difficult therapeutic target, but its commonality to multiple neuro- degenerative conditions makes it an attractive one never- theless. Important questions remain about topics discussed above. It is important to clarify how nuclear-encoded pro- teins reach stationary axonal mitochondria, whether through axonal transport within moving mitochondria, transport as cytoplasmic proteins, or transport of mRNAs encoding them. The answer will help indicate the respec- tive importance of mitochondrial fusion, local protein im- port and synthesis in replenishing the mitochondrial proteome, and may of course differ for different proteins. A better understanding is needed of the wider roles of mitochondrial axonal transport, fusion and fission and how this relates to the many neurodegenerative disorders in which these processes are perturbed. For example, does the failure of increased mitochondrial transport to allevi- ate motor deficits in a mouse model of familial ALS [40] mean that reduced mitochondrial transport in this disor- der is an epiphenomenon or does it reflect subtleties of mitochondrial transport that we do not yet understand? The regulation of mitochondrial quality control, now rea- sonably well understood in cell lines and neuronal soma, needs to be placed in an axonal context where most of the neuronal cytoplasm resides. In WD, there is no indication yet as to how NMNAT activity links to the mPTP activation step, or whether NMNAT exerts its axon protective effect within mitochondria or in an upstream cytoplasmic loca- tion. Fundamental points about mitochondrial NAD + syn- thesis remain unclear, such as whether and how NAD + or its precursor nicotinamide mononucleotide (NMN + ) enters mitochondria. Little is known about how axonal mitochon- dria compare to somatic mitochondria in terms of Ca 2+ handling, ATP production and ROS generation, or about whether moving and stationary mitochondria are equally functional in these regards. It will be important to deter- mine whether intrinsic differences between Wld S and wild type mitochondria underlie axon protection or whether events upstream of the mitochondria determine the differ- ential response to axon injury. In conclusion, axon survival depends on mitochondria both in the short term, to integrate the survival and death response to a wide array of axonal stresses, and in the long

370

term, where quality control requires effective transport, fusion, fission and selective removal of mitochondria. Advances in molecular genetics and live imaging are set to drive further understanding of these critically important processes.

Acknowledgments

Our research is supported by Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT) no. 1110987, Millennium Nucleus no. P07-011- F (to F.C.) and a Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant (to M.C.).

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